RESEARCH ARTICLE
The Effects of Leucine, Zinc, and Chromium
a11111
OPEN ACCESS
Citation: Kolahian S, Sadri H, Shahbazfar AA, Amani
M, Mazadeh A, Mirani M (2015) The Effects of
Leucine, Zinc, and Chromium Supplements on
Inflammatory Events of the Respiratory System in
Type 2 Diabetic Rats. PLoS ONE 10(7): e0133374.
doi:10.1371/journal.pone.0133374
Editor: Dominik Hartl, University of Tübingen,
GERMANY
Received: June 6, 2015
Accepted: June 26, 2015
Published: July 17, 2015
Copyright: © 2015 Kolahian et al. This is an open
access article distributed under the terms of the
Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any
medium, provided the original author and source are
credited.
Data Availability Statement: All relevant data are
within the paper and its Supporting Information files.
Funding: This study was financially supported by the
Tuberculosis and Lung Research Center of Tabriz
University of Medical Sciences and the Research
Council of University of Tabriz. The funders had no
role in study design, data collection and analysis,
decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared
that no competing interests exist.
PLOS ONE | DOI:10.1371/journal.pone.0133374 July 17, 2015
Supplements on Inflammatory Events of the
Respiratory System in Type 2 Diabetic Rats
Saeed Kolahian*1, Hassan Sadri2,3, Amir Ali Shahbazfar4, Morvarid Amani5,
Anis Mazadeh5, Mehdi Mirani5
1 Department of Basic Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran, 2 Institute
of Animal Science, Physiology and Hygiene Unit, University of Bonn, Bonn, Germany, 3 Department of
Clinical Sciences, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran, 4 Department of
Pathobiology, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Iran, 5 Faculty of Veterinary
Medicine, University of Tabriz, Tabriz, Iran
* skolahian@tabrizu.ac.ir
Abstract
Diabetes mellitus is a major cause of serious micro- and macrovascular diseases that affect
nearly every system in the body, including the respiratory system. Non-enzymatic protein
glycation due to hyperglycaemic stress has fundamental implications due to the large capil-
lary network and amount of connective tissue in the lung. The current study was designed
to determine whether leucine, zinc, and chromium supplementations influence the function
and histological structure of the respiratory tract in a rat model of type 2 diabetes. Seventy-
seven rats were divided into eleven groups, consisting of 7 animals each. One group served
as negative control and insulin and glibenclamide were used as positive control drugs.
Thus, eight groups received the nutritional supplements alone or in combination with each
other. Nutritional supplements and glibenclamide were added to the drinking water and neu-
tral protamine Hagedorn insulin was subcutaneously injected during the 4 weeks of treat-
ment period. The induction of type 2 diabetes in the rats caused an infiltration of
mononuclear cells and edema in the submucosa of the trachea and lung, severe fibrosis
around the vessels and airways, and perivascular and peribronchial infiltration of inflamma-
tory cells and fibrin. In the diabetic group, the total inflammation score and Reid index signifi-
cantly increased. Diabetes induction significantly reduced the total antioxidant status and
elevated the lipid peroxidation products in the serum, lung lavage and lung tissue of the dia-
betic animals. Treatment with nutritional supplements significantly decreased the histopath-
ological changes and inflammatory indices in the diabetic animals. Supplementation of
diabetic rats with leucine, zinc, and chromium, alone and in combination, significantly
increased the total antioxidant status and lipid peroxidation level in the diabetic animals.
The nutritional supplements improved the enzymatic antioxidant activity of catalase, gluta-
thione peroxidase, myeloperoxidase, and superoxide dismutase in the diabetic rats. The
present results demonstrate beneficial effects and amelioration of inflammation in the respi-
ratory system of type 2 diabetic rats by leucine, zinc, and chromium supplements, probably
due to their hypoglycaemic and antioxidant properties. Using safe and effective nutritional
1 / 22
 Nutritional Supplements in Diabetic Rat

supplements, such as leucine, chromium and zinc, to replace proven conventional medical
treatments may help to control diabetes and/or its complications.

Introduction
There are limited and contradictory data on the association between diabetes and pulmonary
function. Type 2 diabetes mellitus is the most common endocrine disorder in the world. This
condition is characterized by decreases in insulin secretion, defects in glucose uptake in skeletal
muscle and adipose tissue and increases in glucose production in the liver [1,2]. The latter two
abnormalities are primarily due to insulin resistance [3]. High dietary fat intake is associated
with insulin resistance, and this represents a major risk factor for type 2 diabetes [4]. Diabetes
is known to affect various organ systems, including the kidneys, retina, nerves, and cardiovas-
cular system [5]. Recently, in the context of ongoing research into the pulmonary safety of
inhaled insulin [6–8], the effects of diabetes on pulmonary function has also become a subject
of interest [9–11]. Considering its large vascular network and richness in collagen and elastin,
the pulmonary system is susceptible to undergoing microvascular damage and nonenzymatic
glycation in diabetes. Many authors have investigated the pulmonary function and diffusion
capacity of patients with diabetes, but their findings have been inconsistent [12]. In the present
environment, the use of nutritional supplements has dramatically increased throughout the
world, and major pharmaceutical companies are currently conducting extensive research on
natural materials for their potential medicinal values. From both in vitro and in vivo studies in
humans and laboratory animals, it has become evident that leucine (Leu) functions as a strong
insulin secretagogue [13–15]. Induction of insulin secretion by leucine is mediated by its oxida-
tive decarboxylation, by allosteric activation of glutamate dehydrogenase and by increasing the
oxidation of glutamate [13]. Zinc (Zn), the second most abundant trace element in the body,
plays a role in systemic glycemic control through its effects on insulin biosynthesis within pan-
creatic β-cells and probably by modulating the insulinaemic effects on target tissues [16]. On
the other hand, chromium (Cr) is biologically active as a component of the oligopeptide chro-
modulin (also known as low-molecular-weight Cr-binding substance), which is part of an insu-
lin-signalling pathway [17,18]. It has been shown that chromodulin stimulates both the
tyrosine kinase activity of the insulin-activated insulin receptor and the membrane phospho-
tyrosine phosphatase in insulin-sensitive cells [17,18].
Using nutritional supplements, such as Leu, Zn, and Cr, that are closely associated with
insulin metabolism may provide exciting new information and open up new possibilities for
the prevention or control of insulin resistance and its associated disorders, including respira-
tory system abnormalities. However, to our knowledge, the question of whether supplementing
the diet with Leu, Zn, and Cr affects the respiratory system in patients with type 2 diabetes has
not been addressed. The current study was designed to determine whether Leu, Zn, and Cr sup-
plementation influences the respiratory system in a rat model of type 2 diabetes.

Materials and Methods

Experimental groups
Seventy-seven adult male Wistar rats that were 8 weeks old (mean weight 150–200 g) were
used in this study. The rats in each group were housed separately in single cages placed in a
room with a 12:12-hour light:dark cycle and an ambient temperature of 22 ± 2°C. They were
fed a commercially available food pellet diet (normal diet) and water ad libitum during the

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 Nutritional Supplements in Diabetic Rat

acclimation period (10 days). The blood glucose concentrations for all animals were deter-
mined with a blood glucose monitor (Glucose monitor, Canada) in samples obtained from the
tail vein. All animals showed glucose levels of 80 ± 5 mg/dl. Afterwards, the rats were randomly
categorized into eleven groups of 7 animals each: 1) control (CTR); 2) diabetic (T2D); 3) T2D
+ neutral protamine Hagedorn (NPH) insulin (INS); 4) T2D + glibenclamide (GLC); 5) T2D
+ leucine (Leu); 6) T2D + zinc (Zn); 7) T2D + chromium (Cr); 8) T2D + Leu + Zn (Leu-Zn);
9) T2D + Leu + Cr (Leu-Cr); 10) T2D + Zn + Cr (Zn-Cr); 11) T2D + Leu + Zn + Cr (Leu-Zn-
Cr).
All of the procedures in this study were performed in accordance with the guidelines for the
Care and Use of Laboratory Animals as adopted by the Ethics Committee of the Faculty of Vet-
erinary Medicine of University of Tabriz, Iran (Permit Number: A-12-30511).

Diabetes type 2 induction

The rats in the non-diabetic control group (CTR) were fed a normal chow diet consisting (as a
percentage of total kcal) of 12% fat, 60% carbohydrate, and 28% protein (Javaneh Khorasan
Co., Mashhad, Iran), whereas the diabetic groups were fed a high-fat diet (HFD) consisting of
40% fat, 42% carbohydrate, and 18% protein (Javaneh Khorasan Co., Mashhad, Iran). After
2 weeks of dietary manipulation and an overnight fast, the rats on the HFD were intraperitone-
ally injected with a low dose of streptozotocin (STZ) (35 mg/kg; Sigma-Aldrich, Inc., St. Louis,
Mo., USA) [19]. The animals had free access to food and water after the STZ injection, and
both the STZ-injected and CTR animals continued to receive their original diets for the entire
course of the study.

Diabetes confirmation
Three days after the STZ injection and an overnight fast, the presence of diabetes was verified
by blood glucose concentrations above 250 mg/dl, as determined using a blood glucose monitor
(Glucose monitor, Canada) in samples obtained from the tail veins.

Treatment with nutritional supplements

After confirmation of the induction of diabetes, Leu (AllMax, USA) 15 g/L [13], Zn (Zinc sul-
phate, Alhavi, Iran) 10 mg/L [20] or Cr (Picolinate chromium, Swanson, USA) 5 mg/L [21]
were added to the drinking water of the diabetic rats for 4 weeks. In addition, a subset of the
diabetic rats received NPH insulin (an intermediate-acting insulin; Isophane Lansulin, Exir,
Iran) in a dose of 2 U/day subcutaneously, or glibenclamide (glyburide, Iran Najo, Iran) at
20 mg/L for 4 weeks beginning on the same day as the addition of the nutritional supplements.

Haematological analysis
The animals in all groups were killed by exsanguination at the end of each experimental proto-
col, and blood samples were collected following heart sectioning. The blood samples were
transported and stored in the laboratory where the analyses of the haematological parameters
were performed. The packed cell volume (PCV) was determined by the microhaematocrit
method. The serum total protein and fibrinogen levels were investigated using Biuret and sedi-
mentation methods, respectively. The blood haemoglobin was assessed based on the cyanohae-
moglobin method. The red blood cells (RBCs), white blood cells (WBCs) and differential WBC
counts (lymphocytes, neutrophils, monocytes, eosinophils and basophils) were assessed using
an improved Neubauer haemocytometer.

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 Nutritional Supplements in Diabetic Rat

Measurement of inflammatory cell infiltration

The lungs were lavaged with three separate 2-ml aliquots of phosphate-buffered saline. The
recovered bronchoalveolar lavage fluid was centrifuged (400 g, 10 min), and the pellet was
resuspended in 200 μl of phosphate buffered saline. The cells were counted on a haemocyt-
ometer. The differential cell counts were performed on Cytospin preparations stained with
Giemsa stain (Merck, Darmstadt, Germany) using light microscopy. A total of 200 cells was
counted and classified as neutrophils, eosinophils, or mononuclear cells based on normal mor-
phological criteria.

Histopathology and scoring

The animals in all of the groups were killed by exsanguination at the end of experimental pro-
tocol (4 weeks after diabetes confirmation). The left diaphragmatic lobe of the lung and the tra-
chea were removed and placed into 10% neutral phosphate-buffered formalin. After complete
fixation, the tissues were processed using a tissue processor to pass them through increasing
concentrations of ethanol followed by xylol for tissue dehydration and clearing. Paraffin blocks
were prepared from the tissue samples. The specimens were cut into 4-μm slices and stained
with haematoxylin and eosin (H&E stain). The tissues were examined using a light microscope.
Perivascular and peribronchial inflammation were evaluated by counting the layers of inflam-
matory cells formed around the vessels and airways. At least 20 airways and 20 vessels were
studied in each animal, and the average of the layers was calculated. Tracheal inflammation
and glandular hyperplasia and hypertrophy were measured by calculating the Reid index. The
Reid index is a parameter that is measured as follows: submucosal glandular layer thickness/
tracheal wall thickness from the basement membrane to tracheal cartilage. All of the qualitative
histological changes were recorded.

Biochemical analysis of the samples

Blood was collected into test tubes by cardiac puncture, allowed to clot and centrifuged at
1000 × g to obtain the serum. The lung and tracheal extracts were prepared by snap freezing
the samples in liquid nitrogen, homogenizing with a hand homogenizer and suspending the
homogenates in cold 0.1 M phosphate-buffered saline (pH 7.2). The suspensions were centri-
fuged for 10 min at 4,000 × g.

Lipid peroxidation assay in serum

The malondialdehyde (MDA) levels in the serum were determined using the thiobarbituric
acid test [22], also known as the TBARS assay (thiobarbituric acid reactive substances assay).
To precipitate the serum proteins, 2.5 mL of 20% (w/v) TCA was added to 0.5 mL of the sam-
ple, and the mixture was then centrifuged at 1,500 × g for 10 min. Then, 2.5 mL of 0.05 M sul-
phuric acid and 2 mL of 0.2% thiobarbituric acid (TBA) were added to the pellet, and this
mixture was shaken and incubated for 30 min in a boiling water bath. Then, 4 mL n-butanol
was added and the solution was centrifuged and cooled. Finally, the absorption of the superna-
tant was recorded at 532 nm using a UV/VIS spectrophotometer. A calibration curve was
obtained using a range of concentrations of MDA as a standard to determine the concentration
of TBA—MDA adducts in the samples, and the results were expressed as nmol/dL of serum
[22].

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 Nutritional Supplements in Diabetic Rat

Tissue lipid peroxides

The total lipid peroxidation product, as indicated by MDA formation in the homogenate, was
assayed using the TBA method [23]. Briefly, 2 ml of the TCA—TBA solution (0.67% TBA in
20% TCA) was added to 1 ml of the homogenate, mixed thoroughly and heated for 30 min in a
boiling water bath. After cooling and centrifuging at 1,500 × g for 10 min, the absorbance of
the supernatant was read at 535 nm in a spectrophotometer.

Total antioxidant power assay

The ferric-reducing ability of plasma assay (FRAP), which measures the reduction of the ferric
tripyridyltriazine [Fe(III)–TPTZ] complex to the ferrous tripyridyltriazine [Fe(II)–TPTZ] at
low pH, was used to measure the total antioxidant capacity of the lavages, sera and tissue
homogenates [24]. The Fe(II)–TPTZ complex gives a blue colour with an absorbance maxi-
mum at 593 nm. The results were expressed as equivalents of vitamin C, a potent antioxidant
used for calibration purposes.

Total protein assay

The total protein concentration was measured using the Lowry method based on the reactivity
of the peptide nitrogen[s] with the copper [II] ions under alkaline conditions and the subse-
quent reduction of the Folin-Ciocalteu phosphomolybdic phosphotungstic acid to heteropoly-
molybdenum blue by the copper-catalysed oxidation of aromatic acids [25].

Blood insulin level

The blood insulin levels were measured using a standard commercial ELISA kit (BioMark
Technologies Inc., Canada).

Blood and lung antioxidant enzyme activity levels

The blood and lung antioxidant activity levels were measured using standard methods [26–31].

Measurement of catalase (CAT) activity

CAT was assayed colourimetrically at 620 nm and expressed as the moles of H2O2 consumed/
min/mg protein as described by Sinha (1972). The reaction mixture contained phosphate
buffer (0.01 M, pH 7.0), the tissue homogenate and 2 M H2O2. The reaction was stopped by
the addition of the dichromate:acetic acid reagent (5% potassium dichromate and glacial acetic
acid mixed in a ratio of 1: 3) [26,27].

Measurement of superoxide dismutase (SOD) activity

SOD was measured based on inhibition of the formation of amino blue tetrazolium formazan
in a nicotinamide adenine dinucleotide, phenazine methosulfate and nitroblue tetrazolium
(NADH-PMS-NBT) system, according to method of Kakkar et al. [28]. One unit of enzyme
activity was defined as 50% inhibition of NBT reduction [28].

Measurement of glutathione peroxidase (GPx) activity

GPx activity was assessed by combining the samples with a mixture of 1 U/mL glutathione
reductase and 2 mmol/L glutathione in 1 mL phosphate buffer. The mixtures were preincu-
bated at 37°C for 30 minutes. Subsequently, NADPH and tert-butylhydroperoxide were added,

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 Nutritional Supplements in Diabetic Rat

and the change in absorbance at 340 nm was recorded to calculate GPx activity, as previously
described [29,30].

Measurement of myeloperoxidase (MPO) activity

MPO activity was determined using the o-dianisidine—hydrogen peroxide method. The
change in absorbance was measured at 460 nm (ε 11 300/mol per cm) for 5 min. The results
were expressed as units of MPO/mg protein, where 1 unit of MPO was defined as the quantity
of enzyme that degraded 1 nmol of hydrogen peroxide/min at 25°C [31].

Statistical analysis
In the histopathological data, the differences in the percentage of mucus cells and serous cells,
Reid index, and perivascular and peribronchiolar infiltration of inflammatory cells were com-
pared among the groups using one-way ANOVA. The rest of data were analysed using the
MIXED procedure of SAS 9.2. The treatment was considered to be the fixed effect, and the rat
was considered to be the random effect. The Tukey correction method was used for correction
of the multiple comparisons. The data were expressed as the means ± SEM. The results are pre-
sented in the tables as the least squares means and standard errors of the means. The threshold
of significance was set at P<0.05.

Results
Histopathological changes in the lungs and trachea
Ameliorative effects of nutritional supplements on inflammatory changes in the lungs
and trachea of diabetic rats. Scattered infiltration of small numbers of inflammatory cells
was observed in tracheal submucosa and lung parenchyma of the CTR group. There were no
specific lesions in the respiratory system of this group. In the T2D group, infiltration of mono-
nuclear cells and edema in the submucosa of trachea was observed. Hyperplasia and hypertro-
phy of the submucosal serous glands were obvious (Fig 1). In the lung, alveolar
epithelialization (type II pneumocyte hyperplasia) increases the number of alveolar macro-
phages and leads to remodelling and fibrosis around the vessels and airways, which was more
severe around vessels; perivascular infiltration of fibrin, inflammatory cells and edema; peri-
bronchial infiltration of inflammatory cells; hyperaemia and scattered infiltration of mononu-
clear cells in the lung interstitium; and hypertrophy and hyperplasia of the bronchiolar muscle;
and emphysema was also observed. Treatment with glibenclamide (GLC) decreased the inflam-
mation and submucosal glandular hypertrophy and hyperplasia, perivascular and peribron-
chial edema and inflammation compared with the diabetic group. The extent of emphysema,
increased alveolar macrophage numbers and alveolar epithelialization were less pronounced
than in the T2D group, although they were still noticeable (Fig 2). Treatment with insulin
(INS) decreased the inflammation of the lungs and trachea to a level comparable to all other
treatment groups. The emphysema, edema and epithelialization were reduced compared to the
other groups, and the alveolar macrophages were normal in number. There was no evidence of
hypertrophy or hyperplasia of the bronchiolar muscle. Insulin decreased all of the pathological
effects of diabetes, but the changes did not reach the control level. Leucine slightly decreased
the numbers of alveolar macrophages. The numbers of alveolar macrophages and the inflam-
mation in the lungs and trachea were reduced in the Zn and Cr groups compared to the T2D
group. In the Zn-Cr, Leu-Zn and Leu-Cr groups, the numbers of alveolar macrophages and the
inflammation in the lungs and trachea were less than T2D group. Co-treatment with Zn, Cr

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 Nutritional Supplements in Diabetic Rat

Fig 1. Histopathological findings in the tracheas of rats in all of the experimental groups. A. Control
(CTR): normal architecture of trachea Bar: 125 micrometres; B. Diabetic group (T2D): sub-mucosal gland
hyperplasia. Although most of the glands are serous glands (black arrows), one mucus secretory unit is
observable (white arrow) Bar: 50 micrometres; C. Insulin-treated group (INS): the trachea is approximately
normal. Bar: 125 micrometres; D. Glibenclamide-treated group (GLC): inflammatory cells are obvious in the
lamina propria and submucosa (white arrows). Bar: 12.5 micrometres; E. Leucine-treated group (Leu):
inflammatory mononuclear cells are present in the submucosa (white arrows). Hypertrophic and hyperplastic
glands are present (black arrow) Bar: 50 micrometres; F. Zinc-treated group (Zn): severe inflammation in the
lamina propria and submucosa. Bar: 12.5 micrometres; G. Chromium-treated group (Cr): edema (black
arrows) and inflammation (white arrows) in the lamina propria and submucosa. Bar: 50 micrometres; H. Zinc
and Cr-treated group (Zn-Cr): edema (black arrows) and slight inflammation (white arrows) in the lamina
propria and submucosa. Bar: 12.5 micrometres; I. Leu plus Zn-treated group (Leu-Zn): edema (black arrows)
and slight inflammation (white arrows) in the lamina propria and submucosa. Bar: 12.5 micrometres; J. Leu
plus Cr-treated group (Leu-Cr): inflammation in the submucosa (arrows) Bar: 50 micrometres; K. Zinc plus Cr-
treated group (Zn-Cr): the presence of different types of inflammatory cells in the lamina propria and
submucosa (arrows) Bar: 12.5 micrometre; L. Leucine plus zinc plus chromium-treated group (Leu-Zn-Cr):
slight edema in the submucosa (arrows) Bar: 50 micrometres.
doi:10.1371/journal.pone.0133374.g001

and Leu (Leu-Zn-Cr) markedly decreased the number of alveolar macrophages and the inflam-
mation in the lungs and trachea compared with the T2D group (Figs 1 and 2).
Perivascular and peribronchial inflammation was reduced in the lungs of diabetic rats
after treatment with nutritional supplements. In the T2D group, perivascular cuffing
(PVC), peribronchial cuffing (PBC) and the total inflammation score (the average of PVC and
PBC) were significantly increased compared with the CTR group (P<0.001). Treatment with
INS, but not GLC, significantly decreased the inflammation scores (P<0.001). Treatment with
Cr and Zn significantly decreased inflammation compared with the T2D group (P<0.05 and
P<0.01, respectively). Co-treatment with Leu and Zn effectively reduced the PBC (P<0.05)
and the total inflammation score (P<0.01) compared with the T2D group. Co-treatment with

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 Nutritional Supplements in Diabetic Rat

Fig 2. Histopathological findings in the lungs of rat in all of the experimental groups. Lung
parenchyma. A. Control (CTR): lung normal parenchyma. Bar: 75 micrometres; B. Diabetic group (T2D):
hypertrophy and hyperplasia of the smooth muscles in a secondary bronchiole wall (arrows). Bar: 20
micrometres; C. Diabetic group: severe inflammation around a blood vessel (white arrows) and alveolar
epithelialization (black arrows). Bar: 75 micrometres. D. Insulin-treated group: slight edema (white arrows)
and scattered inflammatory cells (black arrows) around a blood vessel. The lesions are less pronounced than
those of the diabetic group. The alveolar walls are normal with less epithelialization. Bar: 75 micrometres. E.
Leucine-treated group (Leu): increased numbers of intra-alveolar macrophage compared with the control
(arrows). Bar: 75 micrometres. F. Glibenclamide-treated group (GLC): inflammatory cell infiltration around a
secondary bronchiole (white arrow), edema and fibrin accumulation and fibrosis around a vessel (black
arrow). Bar: 75 micrometres. G. Leu-treated group: severe inflammation around a secondary bronchiole
(white arrow) and epithelialization in the alveolar walls. Bar: 75 micrometres. H. Zinc-treated group (Zn):
Peribronchial inflammation (black arrows) and perivascular edema (white arrow). Bar: 75 micrometres. I. Zn-
treated group: hypertrophy and hyperplasia of the smooth muscle in a secondary bronchiole wall (arrows)
Bar: 20 micrometres. J. Cr-treated group: perivascular edema, fibrin accumulation and fibrosis (arrows). Bar:
75 micrometres. K. Chromium-treated group (Cr): alveolar epithelialization (arrows). Bar: 75 micrometre L. Zn
plus Cr-treated group (Zn-Cr): perivascular edema and fibrin accumulation (white arrows) and scattered
infiltration of inflammatory cells (black arrows). Bar: 75 micrometres. M. Leu plus Zn-treated group (Leu-Zn):
perivascular edema and fibrin and collagen accumulation (arrows). Bar: 20 micrometres. N. Leu plus Cr-
treated group (Leu-Cr): severe perivascular inflammation. Bar: 20 micrometres. O. Leu, Zn plus Cr-treated
group (Leu-Zn-Cr): perivascular edema (white arrows) and slight infiltration of inflammatory cells (black
arrows). The alveolar epithelialization is somewhat less than in the diabetic group. Bar: 75 micrometres.
doi:10.1371/journal.pone.0133374.g002

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 Nutritional Supplements in Diabetic Rat

Leu and Cr was not effective on the PVC, PBC and total inflammation scores, and these values
were significantly higher than in the CTR (P<0.001) and INS groups (P<0.01). Chromium
plus Zn, with or without Leu, was able to decrease these three inflammation scores compared
with the T2D group (P<0.05; Fig 3).
The Reid index (RI) decreased in the lungs of the diabetic animals after using nutritional
supplements. The RI was significantly increased in the T2D group compared with the CTR
group (P<0.001). The RI was significantly decreased in the Cr and Zn groups compared with
the T2D group (P<0.05). Leucine alone and or in combination with Zn did not decrease the
RI. Co-administration of Zn and Cr and co-administration of Leu and Cr were not effective on
RI. Co-treatment with Zn, Cr and Leu significantly decreased the RI compared with the T2D
group (P<0.05; Fig 4).

Haematologic analysis
Blood parameter changes in diabetic rats were normalized by nutritional supple-
ments. Diabetes induction significantly increased the PCV and total blood protein, but these
values were decreased in the Leu-Zn and Leu-Zn-Cr groups, respectively (Table 1). The serum
fibrinogen was significantly increased in the T2D group compared with the CTR group. This
increase was significantly attenuated in the Leu-Zn-Cr group (Table 1). The blood RBC

Fig 3. Perivascular, peribronchial inflammation and total inflammation scores in the rat lungs of all of the experimental groups. ***P<0.001,
**P<0.01, *P<0.05 compared with the control. ###P<0.001, ##P <0.01, #P<0.05 compared with the diabetic group. ¶¶¶P<0.001, ¶¶P<0.01, ¶P<0.05
compared with the insulin-treated group. OP<0.05 compared with the chromium-treated group. •P<0.05 compared with the zinc-treated group. □P<0.05
compared with the leucine plus chromium group.
doi:10.1371/journal.pone.0133374.g003

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 Nutritional Supplements in Diabetic Rat

Fig 4. Reid index in the experimental groups. ***P<0.001, *P<0.05 compared with the control. ##P<0.01, #P<0.05 compared with the diabetic group.
CTR = control; T2D = type 2 diabetes; INS = NPH insulin; GLC = glibenclamide; Leu = leucine; Zn = zinc; Cr = chromium.
doi:10.1371/journal.pone.0133374.g004

number and Hb level were significantly increased in the T2D group compared with the CTR
group. Administration of the nutritional supplements slightly reversed the effects of diabetes
induction, but the effects were significant in only some of the treatment groups (Table 1). Dia-
betes induction significantly increased the insulin level in the T2D group compared with the
CTR group. The Leu and Leu-Zn-Cr groups showed slight decreases in blood insulin concen-
trations, but the values were not as low as that of the CTR group (Fig 5). Furthermore, we did
not find any significant differences in WBCs or the differential counts of the WBCs (lympho-
cytes, neutrophils, monocytes, eosinophils, and basophils) among the experimental groups
(Table 1).
Blood antioxidant enzyme activities improved using nutritional supplements in diabetic
rats. The level of catalase activity in the T2D animals was significantly lower than in the CTR
animals (P<0.05). Treatment with INS or GLC had no significant effect on the catalase activity.
Treatment with leu, Leu-Zn, Leu-Cr or Leu-Zn-Cr significantly increased the catalase activity
in the T2D animals (P<0.05; Fig 6A). The glutathione peroxidase activity was significantly
increased in the T2D group compared with the CTR group (P<0.05). The glutathione peroxi-
dase activity was not affected by the INS or GLC treatments. Zn, Leu, leu-Zn, and leu-Zn-Cr
significantly decreased the glutathione peroxidase activity in the diabetic animals (P<0.05; Fig
6B). The myeloperoxidase activity was significantly increased in the T2D group compared with
the CTR group (P<0.05). Treatment with INS or GLC had no significant effect on the myelo-
peroxidase activity, but treatment with any of the nutritional supplements or their

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 Table 1. Haematological parameters of the experimental groups. Data are least squares means and standard error of the mean.

Treatments2

Item1 CTR T2D INS GLC Leu Zn Cr Leu-Zn Leu-Cr Zn-Cr Leu-Zn-Cr SEM P-
Value
PCV (%) 41.2c 48.5a 47.1a 48.5a 43.7bc 45.9a 44.9abc 44.9abc 44.6abc 44.9abc 43.1bc 0.96 <0.001
TP (g/dL) 41.2c 72.5ab 71.9ab 75.3a 65.1bc 67.6abc 66.7abc 64.9bc 66.3abc 65.6abc 67.4abc 2.21 0.002
e abc a ab cde bcde abcd bcde abcde abcd de
FBG (mg/dL) 83.3 325.0 425.0 350.0 135.7 157.1 278.6 192.9 228.6 292.9 114.3 43.7 <0.001
RBC (106/uL) 69.3e 84.5ab 79.7abcd 85.8a 71.0de 79.7abc 72.7cde 78.4abcd 75.9bcde 75.1cde 73.4cde 2.01 <0.001
Hb (g/dL) 138.3d 170.2a 162.3ab 170.5a 146.3cd 157.1abc 152.6bcd 152.9bcd 151.6bcd 151.7bcd 154.4bc 3.48 <0.001
WBC (cells/uL) 9983.3 10975.0 12117 12192.0 10391 11257.0 9320.0 12419.0 10493.0 9881.4 10451.0 954.6 0.38
Neutrophils (%) 17.0 18.8 27.2 26.5 18.7 19.3 18.1 23.0 21.6 22.6 21.6 2.23 0.06
Lymphocytes 78.7 76.3 67 67.5 76.4 75.9 76.0 74.1 73.9 73.7 74.1 2.72 0.14
(%)
Monocytes (%) 2.50 2.17 3.33 4.00 2.14 2.43 2.43 1.57 1.57 1.71 2.43 0.65 0.41
Eosophils (%) 1.28 2.67 2.11 2.63 2.57 2.43 3.29 2.71 3.00 1.71 1.71 0.61 0.46
Basophils (%) 0.32 0.023 0.34 0.015 0.14 0.00 0.14 0.14 0.00 0.29 0.14 0.13 0.45
1

PLOS ONE | DOI:10.1371/journal.pone.0133374 July 17, 2015

PCV = packed cell volume; TP = total protein; FBG = fibrinogen; RBC = red blood cells; Hb = hemoglobin; WBC = white blood cells.
2
CTR = control; T2D = type 2 diabetes; INS = NPH insulin; GLC = glibenclamide; Leu = leucine; Zn = zinc; Cr = chromium.
a–e
Significant differences (P<0.05) among treatments are indicated by different letters.

doi:10.1371/journal.pone.0133374.t001
Nutritional Supplements in Diabetic Rat

11 / 22
 Nutritional Supplements in Diabetic Rat

Fig 5. Blood insulin concentrations in the experimental groups. Different superscript letters (a-d) indicate significant differences among the groups
(P<0.05). CTR = control; T2D = type 2 diabetes; INS = NPH insulin; GLC = glibenclamide; Leu = leucine; Zn = zinc; Cr = chromium.
doi:10.1371/journal.pone.0133374.g005

combinations significantly decreased the myeloperoxidase activity (P<0.05; Fig 6C). The level
of the superoxide dismutase activity in the T2D animals was significantly higher than in those
of the CTR group (P<0.05). Treatment with INS or GLC and with all of the nutritional supple-
ments significantly decreased the superoxide dismutase activity (P<0.05; Fig 6D).

Bronchoalveolar lavage fluid (BALF) analysis

The inflammatory cell infiltration in BALF was slightly reduced by treatment with the
nutritional supplements. As shown in Table 2, diabetes induction increased the total inflam-
matory cell numbers in the BALF. Treatment with INS or GLC increased the inflammatory cell
infiltration into the BALF of the diabetic animals. The nutritional supplements, with the excep-
tion of Zn, decreased the BALF total cell numbers, but this effect did not reach statistical signif-
icance for any of the nutritional supplements groups (Table 2).
Ameliorative effects of nutritional supplements on oxidative stress and protein leakage
in the BALF of the diabetic rats. TBARS measurements (as lipid peroxidation index) and
FRAP (total antioxidant status) were significantly higher and lower, respectively, in the T2D
group compared with the CTR group (P<0.05; Fig 7A and 7B). The treatments, except GLC
and Zn, significantly reduced the TBARs level in the diabetic animals (P<0.05; Fig 7A). Treat-
ment with INS or GLC had no significant effect on the FRAP level in the diabetic animals.
Treatment with Leu, Leu-Zn, Leu-Cr, Zn-Cr and Leu-Zn-Cr significantly increased the FRAP
level in the diabetic animals (P<0.05; Fig 7B). As shown in Fig 8, there was an increase

PLOS ONE | DOI:10.1371/journal.pone.0133374 July 17, 2015 12 / 22

 Nutritional Supplements in Diabetic Rat

Fig 6. Blood antioxidant enzyme activities in the experimental groups. Blood catalase (A), glutathione peroxidase (B), myeloperoxidase (C) and
superoxide dismutase (D) activities was measured in all of the experimental groups. Different superscript letters (a-f) indicate significant differences among
the groups (P<0.05). CTR = control; T2D = type 2 diabetes; INS = NPH insulin; GLC = glibenclamide; Leu = leucine; Zn = zinc; Cr = chromium.
doi:10.1371/journal.pone.0133374.g006

(P<0.05) in the total protein content of the BALF of the T2D animals compared with CTR sub-
jects. Treatment with INS or GLC increased (P<0.05) the protein content of the BALF. Treat-
ment with Leu, Leu-Zn, Leu-Cr or Leu-Zn-Cr slightly reduced the protein leakage in the
diabetic animals, but the changes did not reach the control level (Fig 8).

Lung biochemical analysis

Lung antioxidant enzyme activities improved in the diabetic animals treated with nutri-
tional supplements. The level of catalase activity in the T2D animals was significantly lower
(P<0.05) than that of the CTR subjects. Only treatment with INS significantly (P<0.05)
increased the catalase activity. Treatment with Leu, Leu-Zn, Leu-Cr or Leu-Zn-Cr significantly
increased the catalase activity in the lungs of the diabetic animals (P<0.05; Fig 9A). The gluta-
thione peroxidase activity was significantly (P<0.05) increased in the T2D group compared
with the CTR group. Treatment with INS or with any of the nutritional supplements or their

PLOS ONE | DOI:10.1371/journal.pone.0133374 July 17, 2015 13 / 22

 Table 2. Cell analysis of bronchoalveolar lavage fluid in the experimental groups. Data are least squares means and standard error of the mean.

Treatments2

Item1 CTR T2D INS GLC Leu Zn Cr Leu-Zn Leu-Cr Zn-Cr Leu-Zn-Cr SEM P-Value
c bc abc a bc ab bc bc bc bc bc
Total # 4428.3 5800.0 6165.0 7746.7 4895.0 6354.3 4882.9 5080.0 4805.7 5270.0 4854.0 378.9 <.0001
RBC # 228.3 468.3 278.3 421.7 161.7 454.3 197.1 250.0 268.6 174.3 186.0 78.2 0.06
Non RBC # 4200.0b 4823.3b 5885.0ab 7325.0a 4733.3b 5900.0ab 4678.6b 4540.0b 4530.0b 5095.7b 4668.0b 383.2 <.0001
RBC/HPF 3.00ab 4.83a 2.33ab 3.00ab 2.33ab 4.00ab 1.43b 2.29ab 2.29ab 1.57b 1.61b 0.65 0.02
ab c bc a c ab bc bc bc bc
Lym/HPF 16.0 10.6 12.1 17.9 10.6 16.0 13.6 12.3 13.4 12.7 13.0bc 1.01 <.0001
Epith/HPF 0.50 0.67 0.83 0.67 1.12 0.57 0.57 0.29 0.57 0.71 0.40 0.31 0.88
Macro/HPF 82.8 87.2 85.6 80.0 83.6 82.4 84.4 86.4 85.0 83.4 85.3 1.71 0.22
Goblet cell/HPF 0.15 0.49 0.33 0.51 0.5 0.00 0.29 0.29 0.14 0.43 0.23 0.21 0.80
Neut/HPF 0.66 1.33 1.17 0.83 0.8 1.29 1.14 0.71 1.00 1.00 1.00 0.31 0.91
1
RBC = red blood cells; HPF = high power field; Lym = lymphocyte; Epith = epithelial; Macro = macrophage; Neut = neutrophil; # = number of cell
2
CTR = control; T2D = type 2 diabetes; INS = NPH insulin; GLC = glibenclamide; Leu = leucine; Zn = zinc; Cr = chromium.
a–c
Significant differences (P<0.05) among treatments are indicated by different letters.

doi:10.1371/journal.pone.0133374.t002

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Nutritional Supplements in Diabetic Rat

14 / 22
 Nutritional Supplements in Diabetic Rat

Fig 7. Oxidative stress in the bronchoalveolar lavage fluid (BALF) of the experimental groups. (A) TBARS (Thiobarbituric acid reactive substances)
used as an index of lipid peroxidation and (B) FRAP (Ferric reducing ability of plasma) as an indicator of a total antioxidant status were measured in all of the
experimental groups. Different superscript letters (a-e) indicate significant differences among the groups (P<0.05). CTR = control; T2D = type 2 diabetes;
INS = NPH insulin; GLC = glibenclamide; Leu = leucine; Zn = zinc; Cr = chromium.
doi:10.1371/journal.pone.0133374.g007

Fig 8. Protein leakage in the bronchoalveolar lavage fluid (BALF) of the experimental groups. The total amounts of protein were measured in the BAL
fluid in all of the experimental groups. Different superscript letters (a-d) indicate significant differences among groups (P<0.05). CTR = control; T2D = type 2
diabetes; INS = NPH insulin; GLC = glibenclamide; Leu = leucine; Zn = zinc; Cr = chromium.
doi:10.1371/journal.pone.0133374.g008

PLOS ONE | DOI:10.1371/journal.pone.0133374 July 17, 2015 15 / 22

 Nutritional Supplements in Diabetic Rat

combinations significantly decreased the glutathione peroxidase activity in the diabetic animals
(P<0.05; Fig 9B). The myeloperoxidase activity was significantly (P<0.05) increased in the
T2D group compared with the CTR group. Treatment with INS or GLC had no significant
effect on the myeloperoxidase activity. Treatment with Leu, Leu-Zn, Leu-Cr, Zn-Cr or Leu-Zn-
Cr significantly decreased the myeloperoxidase activity in the diabetic animals (P<0.05; Fig
9C). The level of superoxide dismutase activity in the T2D animals was significantly (P<0.05)
higher than in the CTR animals. Treatment with INS or GLC had no significant effect on the
superoxide dismutase activity in the diabetic animals. Treatment with Leu, Leu-Zn, Leu-Cr,
Zn-Cr or Leu-Zn-Cr significantly decreased the superoxide dismutase activity in the diabetic
animals (P<0.05; Fig 9D).
The lung oxidative stress of diabetic animals was balanced using nutritional supple-
ments. The TBARS and FRAP measurements were significantly higher and lower, respec-
tively, in the T2D group compared with the CTR group (P<0.05; F 10A and 10B). Treatment
with INS or GLC had no significant effect on the TBARS level in the diabetic animals. Treat-
ment with Leu, Leu-Zn, Leu-Cr-Zn, Zn-Cr or Leu-Zn-Cr significantly decreased the TBARS
levels in the diabetic animals (P<0.05; Fig 10A). Except for Zn-Cr, These treatments also sig-
nificantly increased the FRAP level in the diabetic animals (P<0.05; Fig 10B).

Discussion
The present study showed that Leu, Zn, and Cr supplementation influences the function and
histological structure of the respiratory system in a rat model of type 2 diabetes. Mounting evi-
dence over the past few years has suggested that the lung may also be a target organ of diabetes.
In the present study, we found that type 2 diabetes induction in rats resulted in the infiltration
of mononuclear cells and edema in the submucosa of the trachea; hyperplasia and hypertrophy
of the submucosal serous glands in the trachea; lung alveolar epithelialization; severe fibrosis
around the vessels and airways; perivascular infiltration of inflammatory cells and fibrin;
edema; peribronchial infiltration of inflammatory cells; hyperaemia; hypertrophy and hyper-
plasia of the muscles; and emphysema. In the diabetic group, the PVC, PBC, total inflamma-
tion score and Reid index significantly increased. Previous findings showed that
streptozotocin-induced diabetes caused alterations in the ultrastructure of the granular pneu-
mocytes in the inter-alveolar septae [32], the non-ciliated bronchiolar epithelial (Clara) cells
[33] and the collagen and elastin in the alveolar walls [34]. Thickening of the epithelial and cap-
illary basal laminae of the alveoli and centrilobular emphysema were also obvious findings in
autopsies of diabetic subjects [35,36]. Micro-angiopathy in the capillaries of the alveolar septae
and also in the alveolar and pleural arterioles has been shown in diabetic subjects [36]. Treat-
ment with insulin efficiently ameliorated the histopathological disorders caused by diabetes in
these animals. The PVC, PBC and Reid index were also significantly decreased in the INS-
treated animals. In this respect, co-treatment with Zn, Cr and Leu significantly decreased the
respiratory system inflammation and remodelling induced by diabetes and also normalized the
histopathological changes, PVC, PBC and Reid index to levels close to the control level. Hyper-
glycaemia due to diabetes leads to the increased production of free radical intermediates
through the following methods: increased glycolysis, intercellular activation of the sorbitol
(polyol) pathway, auto-oxidation of glucose and non-enzymatic protein glycation [37,38]. It
seems that the observed improvement in the histopathological changes of the respiratory sys-
tem of diabetic animals treated with insulin or nutritional supplements was most likely related
to the glucose-lowering effects of the treatments.
Fibrinogen, a positive acute phase protein, may be elevated in any form of inflammation.
Fibrinogen levels correlate with the degree of pulmonary inflammation in lung diseases and

PLOS ONE | DOI:10.1371/journal.pone.0133374 July 17, 2015 16 / 22

 Nutritional Supplements in Diabetic Rat

Fig 9. Antioxidant enzymes activities in the lungs of the experimental groups. The lung catalase (A), glutathione peroxidase (B), myeloperoxidase (C)
and superoxide dismutase (D) activities were measured in all of the experimental groups. Different superscript letters (a-f) indicate significant differences
among groups (P<0.05). CTR = control; T2D = type 2 diabetes; INS = NPH insulin; GLC = glibenclamide; Leu = leucine; Zn = zinc; Cr = chromium.
doi:10.1371/journal.pone.0133374.g009

may be involved in the increased cardiovascular risk of patients with type 2 diabetes mellitus
[39,40]. In the current study, the increased serum fibrinogen and total protein concentrations
observed in the T2D group were decreased in the animals treated with Leu, Zn, and Cr. In this
context, the increase in the infiltration of inflammatory cells into the BALF in the diabetic rats
provides direct evidence for an acute inflammatory response induced by the diabetes. The
nutritional supplements used in this study decreased these features of inflammation in the dia-
betic animals.
In the present study, diabetes induction significantly reduced the total antioxidant status
and elevated the levels of lipid peroxidation products in the serum, lung lavage and lung tissue
of the T2D animals. The aetiology and pathophysiology of the biological effects of diabetes
mellitus, especially with respect to cell damage, cellular degeneration, and subsequent compli-
cations are mainly attributed to oxidative stress and oxidative stress-induced apoptosis. The
outcome of the long-term complications caused by oxidative stress during chronic diabetes is
substantially influenced by antioxidant capacity status. In our study, the blood and lung

PLOS ONE | DOI:10.1371/journal.pone.0133374 July 17, 2015 17 / 22

 Nutritional Supplements in Diabetic Rat

Fig 10. Oxidative stress in the lungs of the experimental groups. (A) TBARS (Thiobarbituric acid-reactive substances), used as an index of lipid
peroxidation, and (B) FRAP (Ferric reducing ability of plasma), used to indicate the total antioxidant status, were measured in all of the experimental groups.
Different superscript letters (a-c) indicate significant differences among groups (P<0.05).
doi:10.1371/journal.pone.0133374.g010

enzymatic antioxidant activities followed similar trends in the T2D animals. The catalase activ-
ity decreased but the glutathione peroxidase, myeloperoxidase and superoxide dismutase activ-
ities increased compared to the CTR animals. Diseases, including diabetes type 1 or 2, that are
accompanied by increased reactive oxygen metabolite (ROS) synthesis are associated with an
initial compensatory increase in the antioxidant activities, which decrease again during pro-
longed oxidative stress. This effect reflects the depletion of the systemic pool of enzymes and a
reduction in the antioxidant capacity [41,42]. Our findings showed that the selected nutritional
supplements significantly improved the enzymatic antioxidant activities of the blood and lung
to levels close to the control level. Interestingly, the trends of the effects of the treatments with
the nutritional supplement on the enzymatic antioxidant activities of the blood and lung were
found to be similar and showed the same beneficial trend.
It has been shown that Leu reduces oxidative and inflammatory biomarkers and increases
the anti-inflammatory factor in mice and humans [43,44]. In line with our findings, it has been
shown that the peroxidation of lipid molecules induced by ROS, oxidative stress and inflamma-
tion was significantly attenuated with chromium picolinate supplementation [45,46]. Zinc, an
essential microelement, plays special roles in the conducting airways. The localization of the
abundant labile Zn in the apical cytoplasm of the airway epithelium is controlled by specific
zinc transporters [47]. Zinc affects a number of important airway proteins. Zinc increases
ADAM33 (a disintegrin- and metalloproteinase-containing protein) metalloproteinase, which
is important for extracellular matrix changes and airway remodelling after repeated damage
and repair [48]. Zinc enhances agonist binding to the β2 adrenoceptor by cross-linking two
transmembrane domains of this receptor, resulting in positive allosteric modulation and relax-
ation of the airway smooth muscles [49,50]. It has been shown that Zn deficiency is a positive
trigger for the nuclear translocation of Nuclear Factor-kβ (NF-kβ) in rat lungs and human air-
way epithelial cells [51]. NF-kβ is a cytoplasmic transcription factor that, in activated cells,
translocates to the nucleus and mediates expression of many pro-inflammatory cytokines [52].
Zinc has anti-inflammatory, antioxidant and pro-survival effects. Zinc deficiency leads to oxi-
dative damage in the airways by inducing the infiltration of inflammatory cells and increasing
superoxide and nitric oxide production. Zinc deficiency also exacerbates inflammation due to

PLOS ONE | DOI:10.1371/journal.pone.0133374 July 17, 2015 18 / 22

 Nutritional Supplements in Diabetic Rat

acute lung injury or the presence of asthma [53–55]. Taken together, all of these findings and
our results suggest that Zn, Leu and Cr may be useful as potential antioxidant and anti-inflam-
matory dietary supplements for diabetes-induced lung inflammation and oxidative injury.
The insulinotropic effects of Leu have already been documented in previous studies [13–
15]. Zinc is actively involved in insulin biosynthesis within the pancreatic β-cells and may func-
tion to modulate the insulinaemic effects in insulin-dependent tissues [16]. In addition, Cr is
biologically active as a component of chromodulin, which is part of an insulin-signalling path-
way and stimulates the tyrosine kinase activity of the insulin-activated insulin receptor [17,18].
Controlling hyperglycaemia by using dietary supplements, including Leu, Zn, and Cr, may
reduce the hyperglycaemia-induced oxidative injuries in the respiratory tract. The most benefi-
cial effects of the nutritional supplements on the lung functions and histological structure were
observed when they were used together, which may be explained by their additive hypoglycae-
mic effects. Further studies may provide additional clues to the involvement of these supple-
ments in, and the mechanisms underlying, the improved respiratory functions in the subjects
with type 2 diabetes.

Conclusions
Our study demonstrated that the supplementation of diabetic rats with Leu, Zn, and Cr, alone
and in combination, was associated with significant improvements in the function and struc-
ture of respiratory system. The observed improvements may be related to anti-hyperglycaemic
and antioxidant effects of the supplements. Further, supplementation of subjects with type
2 diabetes with these supplements may be an efficient method for preventing and/or treating
diabetes-induced respiratory system dysfunction.

Supporting Information
S1 Checklist. NC3Rs ARRIVE Guidelines Checklist.
(PDF)
S1 Dataset. Raw data of study.
(XLS)

Acknowledgments
The authors express their appreciation to A. Sadeghi for his help in conducting this
experiment.

Author Contributions
Conceived and designed the experiments: SK HS. Performed the experiments: SK HS AAS MA
AM MM. Analyzed the data: SK HS AAS. Contributed reagents/materials/analysis tools: SK
HS AAS. Wrote the paper: SK HS.

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